Transcriptional Silencing of 35S rDNA in Tragopogon porrifolius Correlates with Cytosine Methylation in Sequence-Specific Manner.

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.

Functions of RNAi Pathways in Ribosomal RNA Regulation.

Argonaute proteins, guided by small RNAs, play crucial roles in gene regulation and genome protection through RNA interference (RNAi)-related mechanisms. Ribosomal RNAs (rRNAs), encoded by repeated rDNA units, constitute the core of the ribosome being the most abundant cellular transcripts. rDNA clusters also serve as sources of small RNAs, which are loaded into Argonaute proteins and are able to regulate rDNA itself or affect other gene targets. In this review, we consider the impact of small RNA pathways, specifically siRNAs and piRNAs, on rRNA gene regulation. Data from diverse eukaryotic organisms suggest the potential involvement of small RNAs in various molecular processes related to the rDNA transcription and rRNA fate. Endogenous siRNAs are integral to the chromatin-based silencing of rDNA loci in plants and have been shown to repress rDNA transcription in animals. Small RNAs also play a role in maintaining the integrity of rDNA clusters and may function in the cellular response to rDNA damage. Studies on the impact of RNAi and small RNAs on rRNA provide vast opportunities for future exploration.

Uniparental expression of ribosomal RNA in ×Festulolium grasses: a link between the genome and nucleolar dominance.

Genome or genomic dominance (GD) is a phenomenon observed in hybrids when one parental genome becomes dominant over the other. It is manifested by the replacement of chromatin of the submissive genome by that of the dominant genome and by biased gene expression. Nucleolar dominance (ND) - the functional expression of only one parental set of ribosomal genes in hybrids - is another example of an intragenomic competitive process which, however, concerns ribosomal DNA only. Although GD and ND are relatively well understood, the nature and extent of their potential interdependence is mostly unknown. Here, we ask whether hybrids showing GD also exhibit ND and, if so, whether the dominant genome is the same. To test this, we used hybrids between Festuca and Lolium grasses (Festulolium), and between two Festuca species in which GD has been observed (with Lolium as the dominant genome in Festulolium and F. pratensis in interspecific Festuca hybrids). Using amplicon sequencing of ITS1 and ITS2 of the 45S ribosomal DNA (rDNA) cluster and molecular cytogenetics, we studied the organization and expression of rDNA in leaf tissue in five hybrid combinations, four generations and 31 genotypes [F. pratensis × L. multiflorum (F1, F2, F3, BC1), L. multiflorum × F. pratensis (F1), L. multiflorum × F. glaucescens (F2), L. perenne × F. pratensis (F1), F. glaucescens × F. pratensis (F1)]. We have found that instant ND occurs in Festulolium, where expression of Lolium-type rDNA reached nearly 100% in all F1 hybrids and was maintained through subsequent generations. Therefore, ND and GD in Festulolium are manifested by the same dominant genome (Lolium). We also confirmed the concordance between GD and ND in an interspecific cross between two Festuca species.

New insights on the evolution of nucleolar dominance in newly resynthesized hexaploid wheat Triticum zhukovskyi.

Nucleolar dominance (ND) is a widespread epigenetic phenomenon in hybridizations where nucleolus transcription fails at the nucleolus organizer region (NOR). However, the dynamics of NORs during the formation of Triticum zhukovskyi (GGAu Au Am Am ), another evolutionary branch of allohexaploid wheat, remains poorly understood. Here, we elucidated genetic and epigenetic changes occurring at the NOR loci within the Am , G, and D subgenomes during allopolyploidization by synthesizing hexaploid wheat GGAu Au Am Am and GGAu Au DD. In T. zhukovskyi, Au genome NORs from T. timopheevii (GGAu Au ) were lost, while the second incoming NORs from T. monococcum (Am Am ) were retained. Analysis of the synthesized T. zhukovskyi revealed that rRNA genes from the Am genome were silenced in F1 hybrids (GAu Am ) and remained inactive after genome doubling and subsequent self-pollinations. We observed increased DNA methylation accompanying the inactivation of NORs in the Am genome and found that silencing of NORs in the S1 generation could be reversed by a cytidine methylase inhibitor. Our findings provide insights into the ND process during the evolutionary period of T. zhukovskyi and highlight that inactive rDNA units may serve as a 'first reserve' in the form of R-loops, contributing to the successful evolution of T. zhukovskyi.

Switch them off or not: selective rRNA gene repression in grasses.

Nucleolar dominance (ND) is selective epigenetic silencing of 35-48S rDNA loci. In allopolyploids, it is frequently manifested at the cytogenetic level by the inactivation of nucleolar organiser region(s) (NORs) inherited from one or several evolutionary ancestors. Grasses are ecologically and economically one of the most important land plant groups, which have frequently evolved through hybridisation and polyploidisation events. Here we review common and unique features of ND phenomena in this monocot family from cytogenetic, molecular, and genomic perspectives. We highlight recent advances achieved by using an allotetraploid model grass, Brachypodium hybridum, where ND commonly occurs at a population level, and we cover modern genomic approaches that decipher structural features of core arrays of NORs.

Corrigendum: To Be or Not to Be Expressed: The First Evidence of a Nucleolar Dominance Tissue-Specificity in Brachypodium hybridum.

[This corrects the article DOI: 10.3389/fpls.2021.768347.].

To Be or Not to Be Expressed: The First Evidence of a Nucleolar Dominance Tissue-Specificity in Brachypodium hybridum.

Nucleolar dominance (ND) is an epigenetic, developmentally regulated phenomenon that describes the selective inactivation of 35S rDNA loci derived from one progenitor of a hybrid or allopolyploid. The presence of ND was documented in an allotetraploid grass, Brachypodium hybridum (genome composition DDSS), which is a polyphyletic species that arose from crosses between two putative ancestors that resembled the modern B. distachyon (DD) and B. stacei (SS). In this work, we investigated the developmental stability of ND in B. hybridum genotype 3-7-2 and compared it with the reference genotype ABR113. We addressed the question of whether the ND is established in generative tissues such as pollen mother cells (PMC). We examined condensation of rDNA chromatin by fluorescence in situ hybridization employing state-of-art confocal microscopy. The transcription of rDNA homeologs was determined by reverse-transcription cleaved amplified polymorphic sequence analysis. In ABR113, the ND was stable in all tissues analyzed (primary and adventitious root, leaf, and spikes). In contrast, the 3-7-2 individuals showed a strong upregulation of the S-genome units in adventitious roots but not in other tissues. Microscopic analysis of the 3-7-2 PMCs revealed extensive decondensation of the D-genome loci and their association with the nucleolus in meiosis. As opposed, the S-genome loci were always highly condensed and localized outside the nucleolus. These results indicate that genotype-specific loss of ND in B. hybridum occurs probably after fertilization during developmental processes. This finding supports our view that B. hybridum is an attractive model to study ND in grasses.

Integrating Wheat Nucleolus Structure and Function: Variation in the Wheat Ribosomal RNA and Protein Genes.

We review the coordinated production and integration of the RNA (ribosomal RNA, rRNA) and protein (ribosomal protein, RP) components of wheat cytoplasmic ribosomes in response to changes in genetic constitution, biotic and abiotic stresses. The components examined are highly conserved and identified with reference to model systems such as human, Arabidopsis, and rice, but have sufficient levels of differences in their DNA and amino acid sequences to form fingerprints or gene haplotypes that provide new markers to associate with phenotype variation. Specifically, it is argued that populations of ribosomes within a cell can comprise distinct complements of rRNA and RPs to form units with unique functionalities. The unique functionalities of ribosome populations within a cell can become central in situations of stress where they may preferentially translate mRNAs coding for proteins better suited to contributing to survival of the cell. In model systems where this concept has been developed, the engagement of initiation factors and elongation factors to account for variation in the translation machinery of the cell in response to stresses provided the precedents. The polyploid nature of wheat adds extra variation at each step of the synthesis and assembly of the rRNAs and RPs which can, as a result, potentially enhance its response to changing environments and disease threats.

Personal Perspectives on Plant Ribosomal RNA Genes Research: From Precursor-rRNA to Molecular Evolution.

The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic interactions of rDNA loci, and epigenetic silencing. In this article, we focus on the early rDNA research in plants, performed mainly at the dawn of molecular biology in the 60 to 80-ties of the last century which presented a prequel to the modern genomic era. We discuss - from a personal view - the topics such as synthesis of rRNA precursor (35S pre-rRNA in plants), processing, and the organization of 35S and 5S rDNA. Cloning and sequencing led to the observation that the transcribed and processed regions of the rRNA genes vary enormously, even between populations and species, in comparison with the more conserved regions coding for the mature rRNAs. Epigenetic phenomena and the impact of hybridization and allopolyploidy on rDNA expression and homogenization are discussed. This historical view of scientific progress and achievements sets the scene for the other articles highlighting the immense progress in rDNA research published in this special issue of Frontiers in Plant Science on "Molecular organization, evolution, and function of ribosomal DNA."

The fate of 35S rRNA genes in the allotetraploid grass Brachypodium hybridum.

Nucleolar dominance (ND) consists of the reversible silencing of 35S/45S rDNA loci inherited from one of the ancestors of an allopolyploid. The molecular mechanisms by which one ancestral rDNA set is selected for silencing remain unclear. We applied a combination of molecular (Southern blot hybridization and reverse-transcription cleaved amplified polymorphic sequence analysis), genomic (analysis of variants) and cytogenetic (fluorescence in situ hybridization) approaches to study the structure, expression and epigenetic landscape of 35S rDNA in an allotetraploid grass that exhibits ND, Brachypodium hybridum (genome composition DDSS), and its putative progenitors, Brachypodium distachyon (DD) and Brachypodium stacei (SS). In progenitor genomes, B. stacei showed a higher intragenomic heterogeneity of rDNA compared with B. distachyon. In all studied accessions of B. hybridum, there was a reduction in the copy number of S homoeologues, which was accompanied by their inactive transcriptional status. The involvement of DNA methylation in CG and CHG contexts in the silencing of the S-genome rDNA loci was revealed. In the B. hybridum allotetraploid, ND is stabilized towards the D-genome units, irrespective of the polyphyletic origin of the species, and does not seem to be influenced by homoeologous 35S rDNA ratios and developmental stage.

Regulation of Nucleolar Dominance in Drosophila melanogaster.

In eukaryotic genomes, ribosomal RNA (rRNA) genes exist as tandemly repeated clusters, forming ribosomal DNA (rDNA) loci. Each rDNA locus typically contains hundreds of rRNA genes to meet the high demand of ribosome biogenesis. Nucleolar dominance is a phenomenon whereby individual rDNA loci are entirely silenced or transcribed, and is believed to be a mechanism to control rRNA dosage. Nucleolar dominance was originally noted to occur in interspecies hybrids, and has been shown to occur within a species (i.e, nonhybrid context). However, studying nucleolar dominance within a species has been challenging due to the highly homogenous sequence across rDNA loci. By utilizing single nucleotide polymorphisms between X rDNA and Y rDNA loci in males, as well as sequence variations between two X rDNA loci in females, we conducted a thorough characterization of nucleolar dominance throughout development of Drosophila melanogaster We demonstrate that nucleolar dominance is a developmentally regulated program that occurs in nonhybrid, wild-type D. melanogaster, where Y rDNA dominance is established during male embryogenesis, whereas females normally do not exhibit dominance between two X rDNA loci. By utilizing various chromosomal complements (e.g., X/Y, X/X, X/X/Y) and a chromosome rearrangement, we show that the short arm of the Y chromosome including the Y rDNA likely contains information that instructs the state of nucleolar dominance. Our study begins to reveal the mechanisms underlying the selection of rDNA loci for activation/silencing in nucleolar dominance in the context of nonhybrid D. melanogaster.

Chromosomal inheritance of parental rDNAs distribution pattern detected by FISH in diploid F1 hybrid progeny of Cobitis (Teleostei, Cobitidae) species has non-Mendelian character.

This study was conducted to describe the major and the minor rDNA chromosome distribution in the spined loach Cobitis taenia (2n = 48) and the Danubian loach Cobitis elongatoides (2n = 50), and their laboratory-produced diploid reciprocal F1 hybrid progeny. It was tested by fluorescence in situ hybridisation (FISH) whether the number of 28s and 5s rDNA sites in the karyotypes of diploid hybrids corresponds to the expectations resulting from Mendelian ratio and if nucleolar organiser regions (NOR)were inherited from both parents or nucleolar dominance can be observed in the induced F1 hybrid progeny. Ten (females) or twelve (males) 28s rDNA loci were located in nine uniarm chromosomes of C. taenia. Two of such loci terminally bounded on one acrocentric chromosome were unique and indicated as specific for this species. Large 5s rDNA clusters were located on two acrocentric chromosomes. In C. elongatoides of both sexes, six NOR sites in terminal regions on six meta-submetacentric chromosomes and two 5s rDNA sites on large submetacentrics were detected. The F1 hybrid progeny (2n = 49) was characterised by the intermediate karyotype with the sites of ribosome synthesis on chromosomes inherited from both parents without showing nucleolar dominance. 5s rDNA sites were detected on large submetacentric and two acrocentric chromosomes. The observed number of both 28s and 5s rDNAs signals in F1 diploid Cobitis hybrids was disproportionally inherited from the two parental species, showing inconsistency with the Mendelian ratios. The presented rDNA patterns indicate some marker chromosomes that allow the species of the parental male and female to be recognised in hybrid progeny. The 5s rDNA was found to be a particularly effective diagnostic marker of C. elongatoides to partially discern genomic composition of diploid Cobitis hybrids and presumably allopolyploids resulting from their backcrossing with one of the parental species. Thus, the current study provides insight into the extent of rDNA heredity in Cobitis chromosomes and their cytotaxonomic character.

Nucleolar Dominance in a Tetraploidy Hybrid Lineage Derived From Carassius auratus red var. () × Megalobrama amblycephala ().

Nucleolar dominance is related to the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor's rRNA genes. To investigate nucleolar dominance associated with tetraploidization, we analyzed the changes regarding the genetic traits and expression of 45S rRNA genes in tetraploidy hybrid lineage including F1 allotetraploids (4n = 148) and F2 autotetraploids (4n = 200) derived from the distant hybridization of Carassius auratus red var. (2n = 100) () ×Megalobrama amblycephala (2n = 48) (). Results showed that nucleolar dominance from the females was established in F1 hybrids and it was inherited in F2 hybrids, suggesting that tetraploidization can lead to rapid establishment of nucleolar dominance in the hybrid origin's tetraploid lineage. These results extend the knowledge of nucleolar dominance in polyploidy hybrid animals, which are of significance for the evolution of hybrids in vertebrates.

Structural features of two major nucleolar organizer regions (NORs), Nor-B1 and Nor-B2, and chromosome-specific rRNA gene expression in wheat.

The reference genome sequence of wheat 'Chinese Spring' (CS) is now available (IWGSC RefSeq v1.0), but the core sequences defining the nucleolar organizer regions (NORs) have not been characterized. We estimated that the total copy number of the rDNA units in the wheat genome is 11 160, of which 30.5%, 60.9% and 8.6% are located on Nor-B1 (1B), Nor-B2 (6B) and other NORs, respectively. The total length of the NORs is estimated to be 100 Mb, corresponding to approximately 10% of the unassembled portion of the genome not represented in RefSeq v1.0. Four subtypes (S1-S4) of the rDNA units were identified based on differences within the 3' external transcribed spacer regions in Nor-B1 and Nor-B2, and quantitative PCR indicated locus-specific variation in rDNA subtype contents. Expression analyses of rDNA subtypes revealed that S1 was predominantly expressed and S2 weakly expressed, in contrast to the relative abundance of rDNA subtypes in the wheat genome. These results suggest a regulation mechanism of differential rDNA expression based on sequence differences. S3 expression increased in the ditelosomic lines Dt1BL and Dt6BL, suggesting that S3 is subjected to chromosome-mediated silencing. Structural differences were detected in the regions surrounding the NOR among homoeologous chromosomes of groups 1 and 6. The adjacent regions distal to the major NORs were expanded compared with their homoeologous counterparts, and the gene density of these expanded regions was relatively low. We provide evidence that these regions are likely to be important for autoregulation of the associated major NORs as well as silencing of minor NORs.

Dynamic nucleolar activity in wheat × Aegilops hybrids: evidence of C-genome dominance.

KEY MESSAGE: NOR loci of C-subgenome are dominant in wheat × Aegilops interspecific hybrids, which may have evolutionary implications for wheat group genome dynamics and evolution. After interspecific hybridisation, some genes are often expressed from only one of the progenitor species, shaping subsequent allopolyploid genome evolution processes. A well-known example is nucleolar dominance, i.e. the formation of cell nucleoli from chromosomes of only one parental species. We studied nucleolar organizing regions (NORs) in diploid Aegilops markgrafii (syn: Ae. caudata; CC), Ae. umbellulata (UU), allotetraploids Aegilops cylindrica (CcCcDcDc) and Ae. triuncialis (CtCtUtUt), synthetic interspecific F1 hybrids between these two allotetraploids and bread wheat (Triticum aestivum, AABBDD) and in F3 generation hybrids with genome composition AABBDDCtCtUtUt using silver staining and fluorescence in situ hybridization (FISH). In Ae. markgrafii (CC), NORs of both 1C and 5C or only 5C chromosome pairs were active in different individual cells, while only NORs on 1U chromosomes were active in Ae. umbellulata (UU). Although all 35S rDNA loci of the Ct subgenome (located on 1Ct and 5Ct) were active in Ae. triuncialis, only one pair (occupying either 1Cc or 5Cc) was active in Ae. cylindrica, depending on the genotype studied. These C-genome expression patterns were transmitted to the F1 and F3 generations. Wheat chromosome NOR activity was variable in Ae. triuncialis × T. aestivum F1 seeds, but silenced by the F3 generation. No effect of maternal or paternal cross direction was observed. These results indicate that C-subgenome NOR loci are dominant in wheat × Aegilops interspecific hybrids, which may have evolutionary implications for wheat group genome dynamics and allopolyploid evolution.

Unique nucleolar dominance patterns in distant hybrid lineage derived from Megalobrama Amblycephala × Culter Alburnus.

BACKGROUND: Nucleolar dominance is an epigenetic phenomenon that occurs in interspecific hybrids and involves the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor's rRNA genes. In this paper, changes in the genetics and expression of 45S rRNA genes in F1 and F2 hybrid progeny of blunt snout bream (BSB, Megalobrama amblycephala) × topmouth culter (TC, Culter alburnus) are investigated. RESULTS: The 45S rDNA loci were analyzed by cloning, RT-PCR and sequencing methods. The results show that nucleolar dominance patterns differ in the F1 and F2 hybrids. In the F1 hybrids of BSB × TC, all the tested individuals inherited and expressed the 45S rRNA genes of both BSB and TC, indicating that nucleolar dominance is not established in the F1 hybrids. However, in the F2 hybrids of BSB × TC, five patterns are observed. Pattern 1 inherits and expresses only the 45S rRNA gene of BSB. Pattern 2 inherits the 45S rRNA gene from both BSB and TC, but only expresses the 45S rRNA of BSB. Pattern 3 inherits and expresses the 45S rRNA gene from both BSB and TC. Pattern 4 inherits the 45S rRNA gene from both BSB and TC, but only expresses the 45S rRNA gene of TC. Pattern 5 inherits and expresses only the 45S rRNA gene of TC. CONCLUSIONS: Nucleolar dominance shows distinctive patterns in intergeneric hybrids of BSB × TC. It is not established in F1 hybrids and is random in F2 hybrids. This study provides new insights into the phenomenon of nucleolar dominance in genetic hybrids in vertebrates.

Selective nucleolus organizer inactivation in Arabidopsis is a chromosome position-effect phenomenon.

Nucleolus organizer regions (NORs) are chromosomal loci where hundreds of rRNA genes are clustered. Despite being nearly identical in sequence, specific rRNA genes are selected for silencing during development via choice mechanism(s) that remain unclear. In Arabidopsis thaliana, rRNA gene subtypes that are silenced during development were recently mapped to the NOR on chromosome 2, NOR2, whereas active rRNA genes map to NOR4, on chromosome 4. In a mutant line deficient for ATXR5 or ATXR6-dependent histone H3 lysine 27 (H3K27) monomethylation, we show that millions of base pairs of chromosome 4, including the telomere, TEL4N, and much of NOR4, have been converted to the corresponding sequences of chromosome 2. This genomic change places rRNA genes of NOR2, which are normally silenced, at the position on chromosome 4 where active rRNA genes are normally located. At their new location, NOR2-derived rRNA genes escape silencing, independent of the atxr mutations, indicating that selective rRNA gene silencing is chromosome 2-specific. The chromosome 2 position effect is not explained by the NOR2-associated telomere, TEL2N, which remains linked to the translocated NOR, implicating centromere-proximal sequences in silencing.

Cytomolecular Analysis of Ribosomal DNA Evolution in a Natural Allotetraploid Brachypodium hybridum and Its Putative Ancestors-Dissecting Complex Repetitive Structure of Intergenic Spacers.

Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n = 30), which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n = 10) and B. stacei (2n = 20). Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs) from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic level.

Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus.

Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d-rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p-rDNA dominant progenitor were meiotically unstable, frequently switching to co-dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d-rDNA dominance, indicating immediate suppression of p-homeologs in F1 hybrids. Original p-rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p-rDNA and d-rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co-dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.

Nucleolar dominance and maternal control of 45S rDNA expression.

Using a system of interspecies hybrids, trihybrids, and recombinants with varying proportions of genomes from three distinct Xenopus species, we provide evidence for de novo epigenetic silencing of paternal 45 S ribosomal ribonucleic acid (rRNA) genes and their species-dependent expression dominance that escapes transcriptional inactivation after homologous recombination. The same pattern of imprinting is maintained in the offspring from mothers being genetic males (ZZ) sex-reversed to females, indicating that maternal control of ribosomal deoxyribonucleic acid (rDNA) expression is not sex-chromosome linked. Nucleolar dominance (nucleolus underdevelopment) in Xenopus hybrids appears to be associated with a major non-Mendelian reduction in the number of 45 S rDNA gene copies rather than a specific pattern of their expression. The loss of rRNA gene copies in F1 hybrids was non-random with respect to the parental species, with the transcriptionally dominant variant preferentially removed from hybrid zygotes. This dramatic disruption in the structure and function of 45 S rDNA impacts transcriptome patterns of small nucleolar RNAs and messenger RNAs, with genes from the ribosome and oxidative stress pathways being among the most affected. Unorthodoxies of rDNA inheritance and expression may be interpreted as hallmarks of genetic conflicts between parental genomes, as well as defensive epigenetic mechanisms employed to restore genome integrity.